Nandita Dasgupta et al. / Journal of Pharmacy Research 2012,5(8),4201-4203 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info

Antibacterial Activity of Leaf Extract of Mexican Marigold ( erecta) Against Different Gram Positive and Gram Negative Bacterial Strains

Nandita Dasgupta*, Shivendu Ranjan, Proud Saha, Rahul Jain, Swati Malhotra and M.A. Arabi Mohamed Saleh. School of Bioscience and Technology, VIT University, Vellore – 632014, Tamil Nadu, India Received on:11-05-2012; Revised on: 16-06-2012; Accepted on:28-07-2012

ABSTRACT The antibacterial effect of Mexican marigold (Tagetes erecta) leaf extract at room temperature against 10 gram positive multidrug resistant bacterial isolates including Enterococcus faecalis, Enterococcus faecium. Staphylococcus epidermidis, , Bacillus cereus, Erysipelothrix rhusiopathiae, Propionibacterium acne, Streptococcus pneumoniae, Streptococcus agalactine, Staphylococcus saprophyticus and 6 gram negative multidrug resistant bacterial isolates including , Klebsiella pneumoniae, Pseudomonas auregenosa, Salmonella enteriditis, Acinetobacter baumannii, Alcaligen faecalis were studied by well diffusion method. The maximum antibacterial effect of Mexican Marigold leaf extract among those micro-organism was obtained for Acinetobacter baumannii (Acitvity Index = 0.913333333) and Propionibacterium acne (Acitvity Index = 0.906666667) and minimum was for Streptococcus pneumonia (Activity Index = 0.026666667). The results suggest that species of Mexican marigold i.e. Tagetes erecta has antibacterial effect against airborne causing gram positive and gram negative bacteria and mainly against skin infection causing bacteria, and hence can be useful in developing drugs for like dermatitis, acne, skin races and also can be developed as antiseptic.

Key words: Tagetes erecta, Leaf extract, Gentamicin, Activity index.

INTRODUCTION The Mexican marigold is classified as: Kingdom: Plantae; Phylum: contains sulfur containing ring) may be the active ingredients in Tagetes Angiosperms; Class: ; Order: ; Family: ; species and they have been used against gram negative and gram positive Subfamily: ; Tribe: Tageteae [1]. The tagetes erecta contain 56 bacteria in vitro [15]. Tagetes erecta is also used as interplant with cowpea species of in sunflower family. Tagetes genus is originated for control of nematode pests [16]. Tagetes erecta essential oil also has in North and South America. Now-a-days it is also cultivated in Asian antioxidant properties [17]. countries like India, Bhutan, Nepal, China [2].Use of Tagetes in magical, religious and medicinal uses have been mentioned in history. Tagetes is used MATERIALS AND METHODS in the treatment of hiccups [3]. T.erecta is used in garlands to decorate houses and Idols of God during the festivals in India. Since ancient time parts of this Microorganisms used plant has been used for medicinal purposes and for the skin wash and A total of 10 gram positive and 3 gram negative organisms were used in the dye is used as by Cherokee [4]. Marigold is commonly used in food additives study. The isolates of Enterococcus faecalis, Enterococcus faecium. as a coloring agent and as animal food in fodder (dried flower meal & extract Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, used as supplement for poultry feed). It is used as an ornamental plant and Erysipelothrix rhusiopathiae, Propionibacterium acne, Streptococcus also in tannin or dye industry [5]. It is also used in medicine (folklore) and as pneumoniae, Streptococcus agalactine, Staphylococcus saprophyticus, a poison for non vertebrates and in plant pest control [6]. Marigold has social Escherichia coli, Klebsiella pneumoniae, Pseudomonas auregenosa, value because of its use in religious purposes. The Tagetes is used to adorn Salmonella enteriditis, Acinetobacter baumannii and Alcaligen faecalis were Idols during festivals in India because of its stability [7]. Tagetes erecta is obtained from the culture collection centre of Institute of Microbial used as a food color in the African countries because of its richness in Technology (IMTECH), Chandigarh, India. leutin [8]. The species Tagetes lucida is used to prepare sweet medicinal tea [9]. Tagetes minuta is used to make a popular potato dish called Media Used Ocopa [10]. The essential oil of Tagetes is extracted from its leaves, stems, The media used in the present investigation was Nutrient Agar media, which stalk and flowers by steam distillation [11]. The main components of this oil was obtained from Hi – Media Laboratories Ltd., Mumbai, India. are limonene, ocimene, lagetone and valeric acid [12]. Essential oil of Tagetes can be attributed to its properties like anti biotic, anti microbial, anti parasitic, Antibiotic Gentamycin used anti septic, anti spasmodic [13]. The antibiotic standard used in this investigation was Gentamicin G30 susceptibility test discs (30 mg per disc) which were obtained from Hi – The essential oil is also used to ease phlegm and catarrh, congestions and in Media Laboratories Ltd., Mumbai, India. wound burns, skin diseases, athlete’s foot, colitis and dermatitis [14]. From scientific studies it is observed that thiophenes (natural phytochemicals that Aqueous stock preparations of Mexican marigold leaf Fresh Mexican marigold (Tagetes erecta) leaves were collected from nursery *Corresponding author. at VIT University, Tamil Nadu, India. 50 gm of surface sterilised Mexican Nandita Dasgupta marigold leaves were gently grinded using mortar and pestle. The crude School of Bioscience and Technology, extract were taken and centrifuged at 5000 rpm for 10 minutes at 27°C. VIT University, Vellore – 632014, Tamil Nadu, India

Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4201-4203 Nandita Dasgupta et al. / Journal of Pharmacy Research 2012,5(8),4201-4203 Now the supernatant obtained was the homogenized Mexican marigold leaf extract which was separated in conical flask. The supernatant extracted was SUMMARY AND DISCUSSION used as stock for antibacterial activity and stored in the refrigerator. The activity index of Mexican marigold for different gram positive and gram negative isolated bacterial strain were calculated after antibacterial testing Re – culturing of micro organisms used or Enrichment of culture using agar well diffusion method and antibacterial sensitivity testing with The bacterial cultures were maintained in Peptone Saline Water. Gentamicin. The maximum antibacterial effect of Mexican marigold leaf extract among those micro-organism was obtained for Acinetobacter Analyzing Antibacterial Activity of Mexican marigold (Tagetes erecta) baumannii (Acitvity Index = 0.913333333) and Propionibacterium acne leaf extract using Agar well diffusion assay (Acitvity Index = 0.906666667) and minimum was for Streptococcus 100µl of each bacterial strain was used to make lawn culture on sterile pneumoniae (Activity Index = 0.026666667). Reason for minimum activity Nutrient Agar plates. 6mm wells were made on the plates using cork borer. index on Streptococcus pneumoniae is its capsulated structure. The results To these well 100µl of Marigold leaves extract was added. 100µl of Mexi- suggest us that species of marigold i.e. Tagetes erecta has antibacterial can marigold leaves extract contained 1.05µg of total dry matter. There effect against airborne disease causing gram positive and gram negative should be sufficient distance between the wells and petriplate wall to avoid bacteria and mainly against skin infection causing bacteria, so it can be overlapping of zone of inhibition. The plates were incubated in an upright useful in developing drugs for diseases like dermatitis, acne, skin races and position at 37°C for 24 hours. The diameter of zone of inhibition was also can be developed as antiseptic. measured in millimeter and the results were recorded. The inhibition zones with diameter less than 8mm were not considered as having antibacterial Table 1: Average Activity index of Mexican marigold (Tagetes erecta) activity. for 10 gram positive and 6 gram negative isolated bacterial strains.

Antibiotic sensitivity testing Sr. Name of isolated Average zone Average Zone Average The test microorganisms were also tested for their sensitivity against the No bacterial species of inhibition of inhibition Activity of Tagetes erecta of Gentamicin Index antibiotic (Gentamicin 30 mg). Using sterile cotton swabs, the enriched leaf extract (in mm) cultures were aseptically swabbed on the surface of sterile nutrient agar (in mm) plates. Using ethanol dipped and flamed forceps, the antibiotic (Gentami- cin, 30 mg) disc was aseptically placed over the agar plates sufficiently 1. Enterococcus faecalis 1.123333 1.466666667 0.763333333 2. Enterococcus faecium 1.156667 1.466666667 0.783333333 separated from the wells formed to avoid overlapping of inhibition zone. 3. Staphylococcus epidermidis 0.893333 1.143333333 0.776666667 The plates were incubated at 37°C for 24 hours and the diameter of inhibi- 4. Staphylococcus aureus 1.166667 1.466666667 0.79 tion zones were measured in mm. 5. Bacillus cereus 1.15 1.46 0.773333333 6. Erysipelothrix rhusiopathiae 1.196667 1.476666667 0.806666667 7. Propionibacterium acne 1.34 1.46 0.906666667 Measuring Activity Index 8. Streptococcus pneumoniae 0.006667 0.24 0.026666667 Following formula was used to measure Activity Index, 9. Streptococcus agalactine 0.006667 0.25. 0.037533333 10. Staphylococcus saprophyticus 0.006667 0.14 0.047621428 11. Escherichia coli 1.256667 1.49 0.816666667 Activity Index = (Zone of inhibition of extract/Zone of inhibition of antibi- 12. Klebsiella pneumoniae 0.403333 0.893333333 0.443333333 otic). 13. Pseudomonas auregenosa 1.23 1.406666667 0.87 14. Salmonella enteriditis 0.306667 0.666666667 0.453333333 15. Acinetobacter baumannii 1.196667 1.3 0.913333333 Zone of inhibition of stocks against each bacterial species and similarly 16. Alcaligen faecalis 0.983333 1.166666667 0.84 zone of inhibition of Antibiotic (Gentamicin) were measured.

RESULT AND DISCUSSION

Activity Index of Mexican marigold (Tagetes erecta) leaf extract From the triplicate readings average zone of inhibition for Mexican mari- gold leaf extract, average zone of inhibition for Gentamicin (30 mg) and using this activity index of Mexican marigold leaf extract for 10 gram posi- tive and 6 gram negative isolated bacterial strain were calculated (Table 1).

For gram positive bacterial strains maximum antibacterial effect was found in Acinetobacter baumannii (Acitvity Index = 0.913333333) and minimum for Streptococcus pneumonia (Activity Index = 0.026666667). Also Mexi- can marigold leaf has very less antibacterial effect on Streptococcus agalactine, Staphylococcus saprophyticus having activity index 0.037533333 and 0.047621428 respectively. For other gram positive bacterial strains Mexican marigold has activity index between 0.763 and 9.066.

For gram negative bacterial strains maximum antibacterial effect was found in Acinetobacter baumannii and Pseudomonas auregenosa with the activ- ity index of 0.913333333 and 0.87 respectively, and minimum for Kleb- siella pneumoniae (Activity Index = 0.443333333). Figure 1: Graphical analysis of Activity Index of various isolated gram positive bacterial strain. Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4201-4203 Nandita Dasgupta et al. / Journal of Pharmacy Research 2012,5(8),4201-4203

Figure 10: Zone of inhibition by Figure 11: Zone of inhibition by Mexican marigold on Streptococcus Mexican marigold on Streptococcus pneumonia, zero activity index. agalactine, zero activity index.

REFERENCES 1. Joy, P.P., Thomas, J., Mathew, S., and Skaria, B.P., Ed. Medicinal . Tropical Horticulture Vol. 2. Naya Prokash, Calcutta pub- lisher, 449-632 (2001). 2. Cruz-Ramírez Luís Alfredo et al, Mexican crops of agroalimentary importance, Advances in Agricultural and Food Biotechnology, 35- Figure 2: Graphical analysis of Activity Index of various isolated gram negative 53, (2006) bacterial strain. 3. M. Abdul Halim et al., The use of plants in traditional health care practice of the shaiji community in southwestern Bangladesh, Jour- nal of Tropical Forest Science 19(3): 168–175 (2007) 4. Nilani Packianathan,Saravanan Karumbayaram, Formulation and Evaluation of Herbal Hair Dye: An Ecofriendly Process, journal of pharmaceutical sciences and research, 2 (10), 648-656, (2010) 5. Richard Cantrill, from Tagetes erecta for use in foods for particular nutritional uses, The EFSA Journal, 315 (1), 1-12, (2006) 6. Farjana Nikkon et al., Insecticidal Activity of Flower of Tagetes erecta L. against Tribolium castaneum (Herbst), Research Journal of and Biological Sciences,5(5),748-753,(2009). 7. Kapil V Patil et al., Stability Analysis in Marigold (Tagetes erecta L.) for Flower Yield and Quality Parameters, Research Journal of Agri- cultural Sciences,2(2),237-240,( 2011). 8. D. Jothi, Extraction of natural dyes from african marigold flower Figure 3: Zone of inhibition by mexican Figure 4: Zone of inhibition by mexican (tagetesereecta l) for textile coloration, AUTEX Research Journal, marigold on Erysipelothrix rhusiopathiae. marigold on Propionibacterium acne. 8(2), 49-53,(2008) 9. John M. Maisch, Botanical Medicine Monographs and Sundry on an indigenous species of croton, American Journal of Pharmacy, 57(12), 1-7, (1985) 10. Jose Rafael Lovera, Ed. Food Culture in South America, 1st Edn, Vol 1, Greenwood Publishing Group: 137-200, (2005). 11. Ester R. Chamorro et al., Evaluation of tagetes minuta l. essential oils to control varroa destructor (acari: varroidae), The Journal of the Argentine Chemical Society, 98(1), 39-47 (2011). 12. Mohammad Hadi Meshkatalsadat, Chemical characterization of vola- tile components of tagetes minuta l. cultivated in south west of iran by nano scale injection, Digest Journal of Nanomaterials and Biostructures, 5 (1): 101-106,(2010). Figure 5: Zone of inhibition by Figure 6: Zone of inhibition by mexican marigold on Escherichia coli. mexican marigold on Pseudomonas 13. H. J. D. Dorman, S. G. Deans, Antimicrobial agents from plants: auregenosa. antibacterial activity of plant volatile oils, Journal of Applied Micro- biology, 88(2): 308-316, (2000) 14. Chowdhury M. S. H. et al., Use of plants in healthcare: a traditional ethno-medicinal practice in rural areas of southeastern Bangladesh, International Journal of Biodiversity Science & Management, 5(1): 41-51 (2009) 15. Roberta Piccaglia, Mauro Marotti, Silvia Grandi, Lutein and lutein ester content in different types of and T. erecta, In- dustrial Crops and Products, 8(1) : 45–51(1998). 16. T.I. Olabiyi, E.E.A. Oyedunmade, Marigold (Tagetes Erecta L.) as interplant with cowpea for control of nematode pests, African Crops Science Conference Proceeding, 8(1): 1075-1078, (2007) 17. Rosa Martha Pérez Gutiérrez et al.,, Antioxidant activity of tagetes erecta essential oil, Journal of the Chilean Chemical Society, 51(2): Figure 7: Zone of inhibition by Figure 9: Zone of inhibition by 883-886, (2006). Mexican marigold on Acinetobacter Mexican marigold on Alcaligen baumannii. faecalis. Source of support: Nil, Conflict of interest: None Declared Journal of Pharmacy Research Vol.5 Issue 8.August 2012 4201-4203